Mass spectrometry analysis of proteins interacting with cGAS
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Elite
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture
SUBMITTER: 
Haiyan Gu
LAB HEAD: Qinmiao Sun
PROVIDER: PXD023597 | Pride | 2022-02-07
REPOSITORIES: Pride
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Project description:DNA derived from the genetic material of pathogens or from cellular DNA damage provides a molecular pattern that can be sensed by pattern-recognition receptors of the mammalian innate immune system. In recent years, the cyclic GMP-AMP synthase (cGAS) protein has been characterized as a primary cytosolic DNA sensor during infection with bacteria, DNA viruses, or retroviruses. While the role of cGAS in downstream immune signaling through STING-TBK1-IRF3 proteins is well-defined, regulatory mechanisms of cGAS activity, such as through post-translational modifications (PTMs), are still an active area of research. Here, we report a comprehensive characterization of cGAS phosphorylations and acetylations in three different cell types. Data-dependent proteomic analyses of immunoaffinity purified cGAS was performed in HEK293T cells under control and DNA-challenged conditions (N = 3 each) and human fibroblasts (HFF) cells under control and HSV-1 infected conditions (N = 4 each) to generate candidate cGAS PTM sites. Using parallel reaction monitoring, a total of 11 PTMs (4 phosphorylations and 7 acetylations) were validated in HEK293T, HFF, and THP-1 cells. Of these, 3 phosphorylations and 5 acetylations have not been previously identified. The functions of these modifications were by generating a series of mutants and measuring cGAS-dependent apoptotic and immune signaling activities.
2020-05-11 | PXD017109 | Pride
Project description:The cGAS-STING pathway, a central component of the innate immune system, senses cytosolic DNA and induces interferon-stimulated genes (ISGs) to mediate inflammation. Here we report the unexpected discovery that cGAS senses dysfunctional protein production. Purified ribosomes interact with and stimulate the catalytic activity of recombinant cGAS in vitro. Disruption of the ribosome-associated protein quality control pathway, which detects and resolves ribosome collisions, results in cGAS- and STING-dependent ISG expression, and causes the re-localization of cGAS from the nucleus to the cytosol. Indeed, cGAS preferentially binds collided ribosomes in vitro, and other orthogonal perturbations that lead to elevated levels of collided ribosomes cause re-localization of cGAS as well. Thus, the cGAS-STING pathway senses and responds to translation stress. These findings have implications for the inflammatory responses to viral infection and tumorigenesis, both of which substantially reprogram cellular protein synthesis.
2021-09-09 | PXD019359 | Pride
Project description:The gastrointestinal tract of mammals is inhabited by hundreds of distinct species of commensal microorganisms that exist in a mutualistic relationship with the host. The process by which the commensal microbiota influence the host immune system is poorly understood. We show here that colonization of the small intestine of mice with a single commensal microbe, segmented filamentous bacterium (SFB), is sufficient to induce the appearance of CD4+ T helper cells that produce IL-17 and IL-22 (Th17 cells) in the lamina propria. SFB adhere tightly to the surface of epithelial cells in the terminal ileum of mice with Th17 cells but are absent from mice that have few Th17 cells. Colonization with SFB was correlated with increased expression of genes associated with inflammation, anti-microbial defenses, and tissue repair, and resulted in enhanced resistance to the intestinal pathogen Citrobacter rodentium. Control of Th17 cell differentiation by SFB may thus establish a balance between optimal host defense preparedness and potentially damaging T cell responses. Manipulation of this commensal-regulated pathway may provide new opportunities for enhancing mucosal immunity and treating autoimmune disease. Experiment Overall Design: We compared the gene expression profiles in the terminal ileum of Swiss-Webster GF mice before and after colonization with SFB, which induced robust Th17 cell differentiation. To sieve out host effects, the role of other microbiota, as well as other factors, we also evaluated the transcriptional program induced in Jackson C57BL/6 mice after co-housing with Taconic B6 animals, which also induces Th17 cell differentiation. 0.5 cm of the most distal part of the small intestine was dissected. Total RNA was extracted with TRIzol. RNA was labeled and hybridized to GeneChip Mouse Genome 430 2.0 arrays following the Affymetrix protocols. Data were analyzed in GeneSpring GX10.
2009-11-10 | E-GEOD-18348 | biostudies-arrayexpress
Project description:To elucidate the role of RNF4 in DNA replication and fork reversal, we performed tandem affinity purification (TAP) using a HEK293T cell line that stably expresses SFB-tagged(S-protein tag, Flag epitope tag, and streptavidin-binding peptide tag) wild-type and CS mutant to isolate proteins that associate with RNF4.
2022-08-11 | PXD028397 | Pride
Project description:We have identified ZNF618 as a novel binding partner of UHRF2. We have found that UHRF2 and ZNF618 co-localize at many genomic loci. Examination of genome-wide distribution of SFB-tagged UHRF2 and ZNF618 in 293T cells using ChIP-seq.
2016-05-31 | E-GEOD-80037 | biostudies-arrayexpress
Project description:The hypothesis tested was that lentiviral driven cGAS expression activates antiviral gene expression. Cells were transduced with lentiviruses expressing cGAS or a control (firefly luciferase). Total RNA was harvested 48 h post-transduction and processed for Illumina BeadArray.
2013-12-01 | E-GEOD-52241 | biostudies-arrayexpress
Project description:Microbiota-induced cytokine responses participate in gut homeostasis, but the cytokine balance at steady-state and the role of individual bacterial species in setting the balance remain elusive. Using gnotobiotic mouse models, we provide a systematic analysis of the role of microbiota in the induction of cytokine responses in the normal intestine. Colonization by a whole mouse microbiota orchestrated a broad spectrum of pro-inflammatory (Th1, Th17) and regulatory T cell responses. Unexpectedly, most tested complex microbiota and individual bacteria failed to efficiently stimulate intestinal cytokine responses. A potent cytokine-inducing function was however associated with non-culturable host-specific species, the prototype of which was the Clostridia-related Segmented Filamentous Bacterium, and this bacterial species recapitulated the coordinated maturation of T cell responses induced by the whole mouse microbiota. Our study demonstrates the non-redundant role of microbiota members in the regulation of gut immune homeostasis. Germfree (GF) female 8-9-week-old mice were gavaged twice at a 24-hr interval with 0.5 mL of fresh anaerobic cultures of fecal homogenate from SFB mono-associated mice, fresh feces from Cv mice (Cvd) or from a healthy human donor (Hum). All mice were sacrificed on d8, 20 and 60 post-colonization in parallel to age-matched Cv and GF controls. RNA was extracted from ileal tissue, and processed to biotin-labelled cRNA, and then hybridized to the NuGO array (mouse) NuGO_Mm1a520177. Microarray analysis compared gene expression in ileum tissue of all the treatment groups GF, Cv, Cvd, Hum and SFB (N=3 per treatment group per time-point). Data was considered significant when P<0.05 using the Benjamini and Hochberg false discovery method.
2010-05-05 | E-GEOD-18056 | biostudies-arrayexpress
Project description:To investigate proteins interacting with STING mutants, HEK 293T cells were co-transfected with three STING mutant expression plasmids (human STING WT, STING delCTT (aa343-379 deletion), STING del6 (aa325-379 deletion) in a pcDNA3.1+ backbone) and either pcDNA-cGAS expression plasmid or control empty pcDNA plasmid. Lysed cells were then immunoprecipitated using sheep anti-STING or control sheep anti-IgG antibodies and protein G magnetic Dynabeads.
2024-02-19 | PXD038063 | Pride
Project description:Background ; Rheumatoid arthritis (RA) is a chronic inflammatory disease, characterized by joint destruction and perpetuated by the synovial membrane (SM). In the inflamed SM, activated synovial fibroblasts (SFB) form the major cell type promoting development and progression of the disease by an abnormal expression/secretion of pro-inflammatory cytokines, tissue-degrading enzymes resulting in a predominant degradation of the extra-cellular matrix (ECM), and collagens causing joint fibrosis. We developed a new procedure, based on human knowledge and formal concept analysis (FCA), to simulate and analyze the temporal behaviour of regulatory and signaling networks. It was applied to a regulatory network (containing 18 genes from 5 functional groups) representing ECM formation and destruction in TGFβ - and TNFα -stimulated SFB. Results ; For the modelling of SFB-controlled ECM turnover in rheumatic diseases, Boolean network architecture was used as well as extensive literature information and revision by experimental gene expression data from stimulated SFB. In course of revision, the additional experimental information resulted in different biologically reasonable changes, yielding two Boolean networks that describe TGFβ and TNFα effects, respectively. The final simulations were further analyzed by the attribute exploration algorithm of FCA, integrating again the observed time series in a more fine-grained and automated manner. The generated temporal rules clearly reveal subtle regulatory relationships between different genes, co-expression patterns and converse gene expression regulation in rheumatic diseases. Conclusion ; The developed Boolean network based method for the dynamical analysis of regulatory and signaling networks represents a reliable systems biological solution for the improved understanding of complex regulatory pathways and the interactions among different genes in disease. The resulting knowledge base can be used for further analysis of the ECM system in human fibroblasts and may be queried to predict the functional consequences of observed (e.g. in diseases as RA) or hypothetical (e.g. for therapeutic purposes) gene expression disturbances. Experiment Overall Design: Patients and tissue samples: Experiment Overall Design: Synovial membrane samples were obtained within 10 min following tissue excision upon joint replacement/synovectomy from RA and OA patients (n = 3 each). After removal, tissue samples were frozen and stored at -70°C. Informed patient consent was acquired and the study was approved by the ethics committees of the respective universities. RA patients were classified according to the American College of Rheumatology (ACR) criteria, OA patients according to the respective criteria for osteoarthritis. The preparation of primary semi-transformed synovial fibroblasts from RA and OA patients was performed as previously described (Zimmermann et al., Arthritis Res. 2001;3(1):72-6). Briefly, the tissue samples were minced and digested with trypsin/collagenase P. The resulting single cell suspension was cultured for seven days. Non-adherent cells were removed by medium exchange. SFB were then negatively purified using Dynabeads® M 450 CD14 and subsequently cultured over 4 passages in DMEM containing 100 μg/ml gentamycin, 100 μg/ml penicillin/streptomycin, 20 mM HEPES, and 10% FCS. Experiment Overall Design: Cell stimulation and isolation of total RNA : Experiment Overall Design: At the end of the fourth passage, the SFB were stimulated by 10 ng/ml TGFβ or TNFα in serum-free DMEM for 0, 1, 2, 4, and 12 h. At the end of each time point, medium was removed and the cells were digested with trypsin/versene (0.25%). Following centrifugation and washing with PBS, the cells were lysed with RLT buffer (Qiagen) and frozen at 70°C. Total RNA was isolated using the RNeasy Kit (Qiagen) according to the supplier's recommendation. Experiment Overall Design: Microarray data analysis: Experiment Overall Design: RNA probes were labelled according to the supplier's instructions (Affymetrix®, Santa Clara, CA, USA). Analysis of gene expression was performed using U133 Plus 2.0 RNA microarrays. Hybridization and washing was performed according to the supplier's instructions. Microarrays were analyzed by laser scanning (Hewlett-Packard Gene Scanner). Background-corrected signal intensities were determined using the MAS 5.0 software (Affymetrix®) and normalized among arrays to facilitate comparisons between different patients. For this purpose, arrays were grouped according to patient class the respective stimulus (TGFβ and TNFα, n=6 each). The arrays in each group were normalized using quantile normalization. Experiment Overall Design: See publication for further details.
2009-08-07 | E-GEOD-13837 | biostudies-arrayexpress
Project description:WAC is a known positive regulator of (macro)autophagy. WAC also forms a complex with RNF20/RNF40 to promote H2B monoubiquitination and hence to affect transcriptional regulation. This study addresses whether the WAC/RNF20/RNF40 complex regulates autophagy through effects on gene expression. WAC, RNF20 and RNF40 were knocked-down using pools of siRNAs in HEK293A cells. Each knockdown was in triplicate and the control was RISCfree siRNA. mRNA expression profiles were investigated using an Illumina HT12v4 Bead Array.
2015-12-17 | E-GEOD-66475 | biostudies-arrayexpress